Analysis element and process for its manufacture

ABSTRACT

An analysis element for the determination of an analyte in a liquid sample, especially for medicinal uses. A carrier layer (2) contains, in a reagent domain (4), a reagent applied in a defined pattern by an ink-jet process. The pattern comprises several sets (A, B, C) of compartments (11-20), the compartments (e.g. 11, 13, 15, 17, 19) of the same set (e.g. A) having the same chemical composition, the compartments (11, 13, 15, 17, 19 or 12, 16, 20) of different sets (A or B) containing different reagents and the compartments of different sets being arranged in alteration so that the compartments containing different reagents are close together but nevertheless spatially separated.

This application is a continuation of application Ser. No. 07/736,919filed Jul. 25, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to an analysis element for the determination of ananalyte in a liquid sample, said analysis element having a carrier layerwhich contains, in a reagent domain, a reagent applied in a definedpattern by an ink-jet process.

2. Description of the Prior Art

Analytical examinations on liquid samples, especially body fluids suchas blood or urine, are frequently carried out with the aid of analysiselements, which are also referred to as: solid state analysis elements.They are available in a variety of external forms, especially asextended test strips and as small square sheets. In every case, theyhave one or more test layers which contain the reagents required for theanalysis. The test layers are brought into contact with the liquidsample and the reaction of the analyte with the reagents produces aphysically measurable detection signal, especially a colour change whichcan be measured visually or photometrically. Examples of other knowndetection signals are optical fluorescence, luminescence and, in thecase of electrochemical analysis elements, voltage or current signals.

Particular importance has recently been attached to analysis elementswhich work on the basis of a specific binding reaction between twobioreactive binding partners. Specific binding reactions in this senseare especially immunological interactions, i.e. interactions betweenantigens or haptens on the one hand and antibodies on the other.However, it is also possible to use other specific bioreactiveinteractions such as lectin-sugar, an interaction between an activesubstance and a receptor, the specific binding between biotin andstreptavidin or certain enzyme-substrate binding reactions, e.g.inhibitors or suicide substrates.

The reagents are generally incorporated in the test layers, the normalcase being either that a porous support matrix (e.g. made of paper orplastic) is impregnated with reagent or that, in a layering process, areagent film is produced which contains the reagents dissolved ordispersed in a film former. As a rule, in the manufacture of analysiselements, different mutually incompatible reagents have to beaccommodated so as to be spatially separated. This is conventionallyachieved by joining together (e.g. by welding or adhesive bonding)individual preprepared reagent support elements. These processes arevery expensive, often cause production defects and only allow limitedminiaturization.

It has recently been proposed to use the ink-jet technology originallydeveloped for computer printers (ink-jet printers) in the manufacture ofanalysis elements. In this context, reference may be made to EP-A-119573and EP-A-268237 (U.S. Pat. No. 4,877,745). Both patent specificationscontain more detailed explanations of the previously known state of theart, including ink-jet technology in particular, to which reference ismade here.

The distinguishing feature of the ink-jet technique is that very smallquanta (partial amounts) of a liquid can be applied as drops to acarrier layer with high precision, the precision relating both to theexact positioning of the dot produced by the drop of reagent on thereagent domain and to the reagent volume. The drops can be ejectedsuccessively at high frequency.

Reagent patterns produced by an ink-jet process differ unambiguouslyfrom the patterns obtainable by other printing techniques. Inparticular, comparably fine reagent dots cannot be produced in any otherway with similar uniformity.

A special variant of the ink-jet technique, which is also particularlysuitable for the invention, is the drop-on-demand technique, whereindividual drops of liquid can be produced at any desired point in timeand applied to a carrier layer. In connection with the metering ofbiochemical analytical liquids, especially reagents, only the techniquedescribed in the mentioned patent specifications has so far been used,where the volume of a jet chamber is compressed every time a drop is tobe ejected. A piezoelectric change in the volume of the jet chamber isutilized in particular here. In U.S. patent application entitled "Methodand device for the metered application of a biochemical analyticalliquid to a target" U.S. patent application Ser. No. 07/735,580 filed ofeven date herewith by the present invention now abandoned, the use ofbubble-jet technology for the application of liquid reagents to areagent domain is described which is also suitable for the presentinvention, and such copending application is hereby incorporated byreference. The term ink-jet is to be understood hereafter asencompassing both said procedures.

The ink-jet technique makes it possible to apply reagents to a reagentdomain of an analysis element, with high precision and uniformity, as areagent layer of exceptionally low thickness. Said patent specificationsalso mention the possibility of applying the reagent to the carrierlayer in a particular pattern, e.g. in order to allow a directcomparison between a subdomain coated with reagent and a reagent-freesubdomain, or in order to make the result of the analysis more clearlyvisible, because the formation of colour appears for example in the formof a plus or minus sign.

De-A-27 27 347 and De-C-27 29 233 have disclosed an alternatingarrangement of dots (especially spots) of different reagents which areapplied by a screen printing technique. However, this process is veryexpensive. Moreover, the amounts of reagent applied cannot be meteredaccurately. The dots are relatively large and can only be miniaturizedto a limited extent. Many reagents cannot be processed to pastessuitable for screen printing without being damaged or having theirproperties changed, so this process, which has been know for a longtime, has not achieved any practical significance.

SUMMARY OF THE INVENTION

According to the invention, an analysis element of the type indicated atthe outset is characterized in that the pattern applied to the reagentdomain comprises several sets of compartments, the compartments of thesame set having the same chemical composition, the compartments ofdifferent sets containing different regents and the compartments ofdifferent sets being arranged in alteration so that the compartmentscontaining different reagents are close together but neverthelessspatially separated. The average distance between the outer limits ofthe compartments of different sets is typically less than 1 mm andpreferably less than 0.5 mm. The combination of the measures accordingto the invention is also denoted as "microcompartmentalization"hereafter.

The term "compartment" denotes a delimited sub-domain. A compartment canconsist of one dot or several mutually overlapping dots. Thecompartments which contain different reagents (and thus belong todifferent sets of compartments in the reagent domain) are at least insome cases spatially separated from one another in that they arearranged next to one another (although not necessarily in the sameplane) in the reagent domain, the compartments containing differentreagents being alternate, i.e. compartments of different sets beingalternately adjacent. For practical reasons, it is convenient to haveregular alteration where, for example, the compartments of three sets A,B and C are present in a cyclically repeating pattern A, B, C, A, B, C,A, B etc. In exceptional cases, however, it may also be convenient tohave an alternating pattern without cyclic repetition.

The compartments can also be arranged on top of one another in somecases. A spatial separation of the compartments in the directionperpendicular to their planar dimension (vertical compartmentalization)is possible here if an isolating intermediate layer consisting of asoluble inert isolating substance (e.g. an inert protein or film former)is applied.

In the process according to the invention, the volume of a quantum ofliquid reagent ejected from an ink-jet head is typically between 20 and2000 picoliters and preferably between 100 and 800 picoliters. The areaof the dot produced by such a quantity on the carrier layer is greatlydependent on the properties of the liquid reagent and the carrier layer.It is approximately between 500 μm² and 0.2 mm² and preferably between3000 μm² and 0.1 mm². The quantities of liquid reagent are typicallyejected at a frequency of more than 1000 sec⁻¹ and preferably of between2000 and 20,000 sec⁻¹.

Depending on the test procedure for which an analysis element accordingto the invention is set up, the compartments can contain both elutablereagents and reagents which are solid-phase-bound on the carrier layer,it being possible for the reagent domain to contain exclusivelycompartments with elutable reagents ("elutable compartments"),exclusively compartments with reagents bound to the solid phase ("fixedcompartments") or a mixture of elutable and fixed compartments, thefollowing advantages, inter alia, thereby being achieved according tothe invention.

The reagents in elutable compartments are rapidly dissolved by theliquid sample and mixed together. By the addition ofsolubility-modifying constituents to the liquid reagent, it is possibleto modify the solubility properties of the individual reagents in thecompartments of different sets in order to permit a particular reactionsequence. Also, by varying the layer thickness of differentcompartments, it is possible to influence the dissolution properties soas to permit a flexible adaptation to the reaction sequence in question.Examples of reagents which are used predominantly in elutable form areenzymes, substrates, coenzymes and indicator components, especiallycolour reagents.

Reagents bound to the solid phase can be bound both adsorptively andcovalently. IN the case of adsorptive binding, it is advantageous if (incontrast to the screen printing technique) water-based coating solutionscan be used which do not contain any hydrophobic additives interferingwith the adsorptive binding. Reagents which are to be bound to the fixedphase on the carrier layer can be precisely located, it being possiblein particular to determine the diffusion distances to other reagents(bound to the fixed phase or elutable) in other sets of compartments inaccordance with the requirements of each individual case.

In general, the invention permits very short diffusion distances betweenthe reagents contained in different sets of compartments, and hencerelatively short reaction times and thorough mixing of the reagentswithout special additional measures.

Within the framework of the present invention, it has been establishedthat the microcompartmentalization made possible by ink-jet technologyopens up completely new test procedures.

As already mentioned, the invention is of particular significance forhomogeneous and heterogeneous methods of determination base on thespecific binding capacity of two bioreactive binding partners. In thiscase, at least one set of compartments contains a first binding partnercapable of binding specifically to a second binding partner, which canbe contained in the liquid sample or be a reagent. Often at least one ofthe binding partners is solid-phase-bound on the carrier layer.

In the preparation of fixed compartments, it is frequently advantageousif the binding partner which is to be fixed is applied in aconcentration (per unit area) which is lower than the binding capacity(per unit area) of the surface to which it is fixed. This makes itpossible to avoid the additional process steps, especially removal ofthe excess by washing, which are otherwise necessary in the fixing ofreagents to carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated in greater detail below with the aid ofExamples which are represented schematically in the Figures:

FIG. 1 is an overhead view showing part of an analysis element accordingto the invention,

FIG. 2 is a basic diagram in perspective of the reagent domain of animmunological analysis element, and

FIG. 3 shows an alternative embodiment of the reagent domain of animmunological analysis element.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The reagent domain 1 shown in FIG. 1 contains ten rows of reagentcompartments 11 to 20, which are applied next to one another on acarrier layer 2. Each of the compartments consists of a large number ofdots 3, which are applied to the reagent domain 1 with an ink-jetprinting head. The procedure is preferably as follows:

The carrier layer 2 used is a plastic film (made especially ofpolystyrene) which has first been irradiated with gamma rays understandardized conditions (EP-B1-0 061 167). It is then positioned underan ink-jet printing head which can make precise movements relative tothe carrier layer 2, and is printed with the dots 3.

A special technique is used to achieve a uniform application of thereagent, covering continuously the area of each of the compartments 11to 20. In a first process step, only every other dot within acompartment is applied, the quanta of liquid reagent applied in thisprocess step thus forming spatially separated dots. After the reagenthas been dried (ca 60 sec at room temperature), quanta of liquid reagentare applied in a second step, at a different time, to the spaces betweenthe dots produced in the first step, so as to form a continuouscompartment. This procedure is particularly advantageous when an aqueousliquid reagent has to be applied to a relatively hydrophobic surface ofthe carrier layer 2. Frequently, however, it is also possible to avoidthe need for the described two-step procedure by influencing the surfaceproperties of the carrier layer material or by modifying the compositionof the liquid reagent.

In the case illustrated, the compartments 11 to 20 are identical intheir external form and in their physical structure (which is preferredalthough not necessary), but differ in respect of their chemicalcomposition. They can be divided into sets, which are denoted by theletters A, B and C in FIG. 1. The compartments within a set contain thesame reagent composition, while the compartments of different setsdiffer in respect of their reagent composition, the compartments ofdifferent sets being arranged alternately so that the compartmentscontaining different reagents are close together but neverthelessspatially separated.

In the case illustrated, every other compartment is to be assigned toset A. The number of compartments in each of sets B and C is only halfas large and they are placed alternately in the gaps between thecompartments of set A.

The dimensions of the compartments and the distances between them areexceptionally small. In one case evaluated in practice, the distancebetween the dots within one compartment was only about 0.14 mm, thecentre-to-centre distance between the compartments was about 0.26 mm inthis example and the average distance between the limits of thecompartments was less than 0.15 mm.

The compartments of different sets can advantageously be produced in asingle pass with the aid of a multichannel printing head or a printerequipped with several printing heads. Printers of these types, workingby the ink-jet process, were developed for colour printing. The patternof compartments shown in FIG. 1 can be produced for example by usinglinearly arranged jets of a multichannel printing head for the differentcompartments and moving the printing head over the carrier layer 2 inthe direction perpendicular to the linear arrangement of jets (arrow 7).This makes it possible to manufacture the analysis elements according tothe invention in a precise and at the same time economic manner.

The movement of the printing head relative to the carrier layer can beeffected with the constructions conventionally used for ink-jetprinters. Within the framework of the invention, it is advantageous tooperate the printing head unidirectionally in order to permitparticularly precise positioning of the dots.

FIG. 2 is a very schematic basic diagram showing the layer structure inthe reagent domain of an analysis element for immunologicaldeterminations.

Three sets of compartments--A (compartments 11, 13, 15, 17 and 19),B(compartments 12 and 16) and C (compartments 14 and 18)--are againshown on the carrier layer 2. The three-dimensional representationreveals another preferred measure, namely that the individualcompartments are separated from one another by a separating layer 5containing an inert water-soluble substance, in particular awater-soluble protein such as bovine serum albumin. The separating layeris applied by a procedure in which, after application of thecompartments belonging to set A, the BSA is applied in several passesfrom a printing head, not only the jets directed at compartments 11 to20 but also intermediate jets of the pressure head being activated inorder to form the continuous BSA layer.

The compartments 11 to 19 and the BSA separating layer 5 together form areagent layer 4 on the carrier layer 2. In practice, the shape of thecompartments is not as uniform and rectangular as shown in FIG. 2. It isa characteristic of the invention, however, that the compartments arespatially separated from one another, at least some of the compartmentsbelonging to different sets being arranged side by side on the carrierlayer 2 (horizontal compartimentalization).

In a preferred embodiment, the compartments of set A contain a firstbinding partner fixed to the support, Bp(b) (binding partner, bound). Itis advantageous here if the compartments containing the first bindingpartner are covered with a protective layer 5a of an inert protectingsubstance. A soluble protein which does not react with the other testcomponents is particularly suitable. It is often mixed with a sugarcompound. The protective layer ensures the required storage stability ofthe binding partner fixed to the support, BP(b).

At least one other set of compartments B or C contains a second, freebinding partner, Bp(f) (binding partner, free), capable of bindingspecifically to Bp(b), "free" meaning that it can easily be dissolved bythe liquid sample. Bp(f) is preferably labelled by a methodconventionally used in immunology (for example by conjugation with alabelling substance M, e.g. an enzyme or a fluorescent marker). In thiscase, as well as with other elutable compartments, it is advantageous ifa blocking portion 5b, covering the carrier layer 2, is arranged underthe compartments 12, 16. The blocking portion 5b is also convenientlybased on a soluble inert protein and serves to prevent unspecificbinding of the elutable reagent (Bp(s) here) to the carrier layer 2 andhence to improve the elutability of Bp(f).

In the embodiment shown in FIG. 2, the protective portions 5a, whichcover the compartments 11, 13, 15, 17 and 19, and the blocking portions5b, which are arranged underneath the compartments 12, 14, 16 and 18,run into one another and together form the separating layer 5. This isparticularly advantageous, although not necessary. Provision could alsobe made for separated protective and blocking portions.

Further details depend on the immunological test principle on which theanalysis element works. For example, if the analyte is an antigen Ag(s)(antigen, sample, Bp(f) can be an antigen Ag(f) analogous to Ag(s) whenusing a competitive test principle. Bp(b) in this case is an antibodyAb(b) (antibody bound) capable of binding specifically to Ag(f). Theanalytical procedure here is based on the fact that Ag(s) competes withAg(f) for binding sites on Ab(b), the amount of Ag(f) bound to Ab(b),which is detectable due to the labelling of Ag(f), being a measure ofthe concentration of the analyte. Via the labelling, the concentrationof Ag(f) can be determined preferably in the bound phase, but inprinciple also in the free phase.

According to another immunological reaction principle ("one-stepsandwich"), the compartments B or C (again for determining an antigenAg(s) can contain an antibody Ab(f) capable of binding specifically toAg(s). In this case, the set of compartments A contains an antibodyAb(b) fixed to the support, which is capable of binding specifically toAg(s) via a second epitope. The test method is based in this case on thefact that Ag(s) promotes binding between Ab(b) and Ab(f).

If an antibody is to be determined rather than an antigen, the bound andfree immunological reaction components have to be respectively exchangedin the compartments (antigen for antibody and vice-versa).

These and other immunological test principles suitable for the inventionhave been known for a long time. Reference may be made, for example, toU.S. Pat. No. 4,861,711 and numerous other publications describing theapplication of heterogeneous immunological reactions to analysiselements in different variants.

Table 1 shows the arrangement of the sets of compartments for theaforementioned reaction principles as well as a few others:

                  TABLE 1                                                         ______________________________________                                                          Bp(b)   Bp(f) in                                                    Bp(s)     in A    B and/or C                                          ______________________________________                                        sandwich  Ag          Ab      Ab-M                                                      Ab          Ag      Ag-M                                            competitive                                                                             Ag          Ab      Ag-M                                                      Ab          Ag      Ab-M                                            ______________________________________                                    

compared with the known immunochemical analysis elements, the inventionachieves a significant simplification of the manufacture and structureof an imunological analysis element by applying the ink-jet technique toproduce compartments of different immunological reaction componentsarranged in alternation and spatially separated, but nevertheless closetogether.

It is found that the soluble binding partner contained in a first set ofcompartments is very rapidly dissolved by the sample, so the reaction ofthis binding partner with the analyte begins immediately after contactwith the sample. At the same time, the entire sample is in contact withthe binding partner fixed to the support. The microcompartmentalizationof the reagents enables the binding reactions in question to proceedrapidly and homogeneously with a very small amount of sample and reagentand a high reaction rate. Here the reaction with the free bindingpartner preferably takes place first, while the reaction with thesolid-phase-bound binding partner proceeds substantially more slowly asa heterogeneous reaction and, in practice, does not set in to asignificant extent until after the binding reaction of Bp(f) has takenplace.

According to a preferred embodiment of the invention, at least threedifferent sets of compartments are applied to the reagent domain of ananalysis element: a first binding partner Bp(b), fixed to the support,in Set A, a second binding partner Bp(f)2, which is free and carries amarker, in set C and a third binding partner Bp(f)1, which is also free,in Set B. Here the third binding partner Bp(f)1 is capable of bindingboth to the first binding partner Bp(b) and to the second bindingpartner Bp(f)2 with different specificities. Preferably, the firstbinding partner Bp(b) is the same for different analysis elements, whilethe two free binding partners are selected according to the analyte(parameter) to be determined and the chosen method. The first bindingpartner preferably contains streptavidin (SA) or avidin and the thirdbinding partner preferably contains biotin (b). The biotin is conjugatedwith an antigen or antibody, depending on the test procedure, to giveAg-B or Ab-B.

Table 2 shows the arrangement of the sets of compartments for twodifferent test principles, it being assumed in each case that an antigenis to be determined in the sample. If an antibody is to be determined,antigen and antibody have to be exchanged.

                  TABLE 2                                                         ______________________________________                                        To determine an Ag                                                                          Bp(b)      Bp(f)1  Bp(f)2                                       ______________________________________                                        competitive (a)                                                                             SA         B-Ag    Ab-M                                         (b)           SA         B-Ab    Ag-M                                         sandwich      SA         B-Ab    Ab-M                                         ______________________________________                                    

With each of these principles, after dissolution of the free bindingpartners, Bp(f)1 binds to Bp(b) via the avidin-biotin bond.

In the competitive test of type (a), the binding of Ab-M to B-Ag isdetermined by competition with the sample antigen Ag(s). In thecompetitive test of type (b), Ag(s) competes with Ag-M for binding siteson the antibody of B-Ab. In the sandwich test, the sample antigen againpromotes binding between the antibody of B-Ab and the antibody of Ab-M.

In this embodiment, the streptavidin (or avidin), which is fixed to thecarrier, and the biotin, which is covalently bound to other reagents,form a so-called capturing system. The use of a capturing system makesit possible easily to bind different reagent components to a fixed phasewhich has been pretreated homogeneously (with a single bindingcomponent, in this case streptavidin). Within the framework of thepresent invention, this can furthermore be done in a precisely locatableform. On the other hand, it is also possible to apply solublecompartments (non-biotinylated reagents) to a carrier layer which hasbeen pretreated homogeneously, for example with streptavidin, withouttheir solubility properties being substantially affected. Furtherdetails can be found in EP-A-0 269 092 and EP-A-0 344 578.

FIG. 3 shows an alternative embodiment of a test carrier with three setsof compartments containing three different binding partners. The testcomposition is essentially the same as that of the previous embodiment,except that in this case the third binding partner is coated directly onthe first binding partner and is thus bound to the latter. The two boundbinding partners are therefore denoted as Bp(b)1 and Bp(b)2. They arelocated in double compartments 20, 22, 24, 26, 28 of set A. The secondbinding partner is a free binding partner Bp(f) in the compartments 21,23, 25, 27 of set B.

The following Examples serve to illustrate the invention further. Unlessstated otherwise, all data in % denote percentages by weight.

EXAMPLE 1

an Epsom SQ-2550 ink-jet printer was used to apply the compartments. Inplace of the ink reservoir, a separate reagent reservoir, containing theparticular reagent solution to be metered, was connected to the systemof tubes leading to the printing head. The printer was run in thegraphics mode (individual jet drive) via a personal computer.

The support material used was a 0.1 mm thick DIN A4 blank made ofpolystyrene film. The film was subjected to standardized gammairradiation before use.

Characteristics of the printing head:

24 jets arranged in two rows (offset by half a line)

drop diameter: ca. 90 μm

smallest meterable volume (1 drop): ca. 420 picoliters

print density per printing step: 180×180 dots/inch2.

The compartments are applied by the procedure described in connectionwith FIG. 1. Each of the reagent compartments consists of 24 individualdrops of ca. 400 picoliters each. The dimensions of a compartment areca. 3.2 mm high by ca. 0.06 to 0.08 mm wide. The centre-to-centredistance between the compartments is ca. 0.26 mm.

The separating layer based on bovine serum albumin ("BSA separatinglayer" hereafter) was applied with a total of four print runs, thedistance between dots being 0.14 mm horizontally and 0.14 mm vertically.

The arrangement of the compartments in the reagent domain correspondedto FIG. 2. The following solutions were used for the individual sets ofcompartments.

a) Set of compartments A: Bp(b) (namely SA): SA fixed to the support

    ______________________________________                                        Coating solution:                                                                            0.25 mg/ml                                                                              TBSA-SA                                                            40 Mm      sodium phosphate                                                              buffer (NaPB) pH 7.4                                                5 vol %   isopropanol                                          ______________________________________                                    

TBSA-SA denotes thermo bovine serum albumin-streptavidin according toEP-A-0 269 092 and EP-A-0 344 578. This compound is especially suitablefor the adsorptive fixing of streptavidin to the support. The amount ofTBSA-SA applied per unit area was marginally less than the adsorptivebinding capacity of the support film, thereby avoiding additionalprocess steps to ensure quantitative and stable binding to the fixedphase.

b) Protective layer 5: BSA separating layer

    ______________________________________                                        Coating solution:                                                                         0.6%      bovine serum albumin (BSA)                                          4.0%      sucrose                                                             1.8%      sodium chloride (NaCl)                                              5.0 vol % isopropanol                                             ______________________________________                                    

c) Set of compartments B: Bp(f)1 (namely B-Ag): Elutable conjugateconsisting of T3 and biotin (T3-biotin), prepared by coupling biotinwith N-butoxycarbonyltriiodothyronine (T3) via pentamethylenediamine(Eur. J. Biochem, 131 (1980, 333-338)).

    ______________________________________                                        Coating solution:                                                                         200 ng/ml  T3-biotin                                                          120 mM     sodium barbiturate                                                 21.8 mM    NaPB pH 8.35                                                       0.04%      8-anilinonaphthalene-                                                         1-sulphonic acid (ANS)                                             0.02%      4-aminoantipyrine                                                  0.01%      Merthiolate                                                        0.25%      bovine IgG (B-IgG)                                                 1.0%       Pluronice<< F68                                        ______________________________________                                    

d) Set of compartments C: Bp(f)2 (namely Ab-M): Elutable conjugateconsisting of a polyclonal anti-body directed against T3 and peroxidase(PAB<T3>-POD).

    ______________________________________                                        Coating solution:                                                                           22.2 U/ml  PAB<T3>-POD                                                        120 mM     sodium barbiturate                                                 21.8 mM    NaPB pH 8.35                                                        0.04%     ANS                                                                 0.02%     4-aminoantipyrine                                                   0.01%     Merthiolate                                                         0.25      B-IGG                                                               1.0%      Pluronice<< F68                                      ______________________________________                                    

Test strips of 3.2 mm in height and 15 mm in length were cut out of thecoated polystyrene film, the compartments running over their entireheight perpendicularly to the longitudinal direction. The total molaramounts of reagent present in such a test strip in the particularcompartments were:

In the set of compartments C: 0.05 femtomol of PAB<T3>-POD (=1.4 μU)

In the set of compartments B: 23.0 femtomol of T3-biotin

In the set of compartments A: ca. 500.0 femtomol of biotin binding sites(through TBSA-SA)

A T3 analysis was carried out as follows:

50 μl of each T3-containing sample were applied to a test strip andincubation was carried out for 2.5 h at room temperature in a humiditychamber.

The test strip was washed 4 times with 1 ml of the BSA solution used forthe protective layer.

A substrate for the labelling enzyme, namely 50 μl of diaminobenzidine(DAB) colour reagent (0.5 mg/ml of DAB, 40 mM NaPB pH 7.4, 0.025% ofcobalt chloride, 0.02% of nickel sulphate, 0.01% of hydrogen peroxide),was applied and incubation was carried out for one hour at roomtemperature in the humidity chamber.

The strip was then washed with water.

The test is based on the competitive principle explained above: the T3competes with the biotinylated T3 for binding sites on the PAB<T3>-PODconjugate.

A visually recognizable colouration of the set of compartments A, ofvarying intensity, was produced as a function of the T3 concentration inthe sample. Determination of the pattern of compartments by ameasurement technique permits calibration and quantitative evaluation.

EXAMPLE 2

Construction and manufacturing process corresponding to Example 1 withthe exception of the composition of the solutions used for the sets ofcompartments B and C. These had the following compositions:

a) Set of compartments B: Bp(f)1 (namely B-Ab): Elutable conjugateconsisting of a monoclonal antibody directed against TSH (ECACC87122201) and biotin (MAB<TSH>-biotin). The biotinylation of theantibody was carried out according to JACS 100 (1978, 3585-3590) byreaction with N-hydroxysuccinimidobiotin in a ratio of 10:1.

    ______________________________________                                        Coating solution:                                                                           80 Mm     NaPB pH 7.4                                                         65 μg/ml                                                                             MAB<TSH>-biotin                                                     0.6%      BSA                                                                 0.3%      bovine IgG                                            ______________________________________                                    

b) Set of compartments C: Bp(f)2 (namely Ab-E): Elutable conjugateconsisting of peroxidase and a monoclonal antibody directed against TSH(ECACC 87122202) (MAB<TSH>-POD).

    ______________________________________                                        Coating solution:                                                                           50 mM     NaPB pH 7.4                                                          9 U/ml   MAB<TSH>-POD                                          ______________________________________                                    

The test procedure corresponds to the heterogeneous one-step sandwichtest with streptavidin-biotin capturing system for binding of theimmunological complex to the fixed phase.

Test strips were again prepared with the same dimensions as inExample 1. Each test strip comprises 18 compartments of set A and 9compartments each of sets B and C.

The analysis was performed as in Example 1, except that in the firststep 100 μl of each TSH-containing sample were applied and incubationwas carried out for 3 h at RT in a humidity chamber.

As in Example 1, a visually clearly recognizable colouration, of varyingintensity, of the compartments of set A was produced.

We claim:
 1. An analysis element for bioreactively analyzing a liquidsample, said analysis element comprising:a carrier layer having aplurality of sets of compartments thereupon, each of said compartmentscomprising a reagent applied in a predetermined pattern by an ink-jetprocess, the compartments of a first set being fixed compartmentscontaining a first binding partner which is solid phase bound to thecarrier layer, and which is capable of binding specifically to acorresponding binding partner which is contained in the liquid sample orin a second set of compartments, said fixed compartments containing thefirst binding partner being covered by a layer of an inert water solubleprotein substance, with compartments of at least one set being elutablecompartments containing a labelled second binding partner which iselutable and which is disposed adjacent to a top surface of the layer ofinert water soluble protein substance, said labelled second bindingpartner being capable of binding bioreactively and specifically to acorresponding binding partner contained in the liquid sample or anotherset of compartments, wherein the layer of inert water soluble proteinsubstance is located between the carrier layer and the elutablecompartments containing the labelled second binding partner, and overthe fixed compartments, wherein the layer of inert water soluble proteinsubstance forms a continuous layer which spatially separates said fixedcompartments and said elutable compartments, wherein said fixedcompartments and said elutable compartments are arranged in analternating horizontal relationship with the layer of inert watersoluble protein substance therebetween.
 2. The analysis element of claim1, wherein the first binding partner is bound to the carrier layer in aconcentration which is lower than a binding capacity of the carrierlayer.
 3. The analysis element of claim 1, wherein a third bindingpartner is contained in a third set of compartments, said third set ofcompartments being disposed adjacent to said second set of compartments,wherein the third binding partner is a conjugate which comprises a firstcomponent with binding affinity for said first binding partner and asecond component with binding affinity for either said second bindingpartner or a corresponding binding partner in the liquid sample.
 4. Theanalysis element of claim 3, wherein the first binding partner comprisesavidin or streptavidin and the first component of the third bindingpartner is biotin.
 5. The analysis element of claim 3, wherein the firstbinding partner and the third binding partner form a capturing system.6. The analysis element of claim 1, wherein said carrier layer is aplastic layer.
 7. A process for manufacturing an analysis element forbioreactively analyzing a liquid sample, said process comprising thesteps of:providing a carrier layer; applying a plurality of discretequantities of a liquid reagent comprising a first binding partner toform separate compartments on said carrier layer by ejecting the liquidreagent in discrete droplet form from an ink-jet head onto a reagentdomain portion of the carrier layer; applying a layer of inert watersoluble protein substance to said reagent domain portion of the carrierlayer, thereby covering said discrete quantities of liquid reagents andportions of said carrier layer with no applied liquid reagent; andapplying a labelled second binding partner id discrete droplet form froman ink-jet head upon selected portions of said layer of inert watersoluble protein substance; wherein the ink-jet head and carrier layerare moved relative to each other during said applying steps in apredetermined pattern so that the dots produced on the carrier layer bythe droplets form a plurality of the compartments on the reagent domainportion, with the compartments of a first set being fixed compartmentscontaining the first binding partner which is solid phase bound to saidcarrier layer, and which is capable of binding specifically to a secondbinding partner in said liquid sample or in said second set ofcompartments, with compartments of at least one second set beingelutable compartments containing the second binding partner which isdisposed adjacent to a top surface of the layer of inert water solubleprotein substance, said second binding partner being elutable and whichis capable of binding specifically to a corresponding binding partnercontained in the liquid sample or the first set of compartments, andwherein the layer of inert water soluble protein substance is locatedbetween the carrier layer and the elutable compartments containing thesecond binding partner, and over the fixed compartments which forms acontinuous layer that spatially separates said fixed compartments fromsaid elutable compartments, with the fixed compartments and the elutablecompartments being arranged in an alternating horizontal relationshipwith the layer of inert water soluble protein substance therebetween. 8.The process of claim 7, wherein the carrier layer is a plastic layer,and including the further step of irradiating the plastic layer withgamma rays prior to application of the liquid reagents.
 9. The processof claim 7, wherein the droplets of one of said liquid reagents areejected to form a series of spatially separated first dots on thereagent domain as said separate compartments, and wherein a plurality ofdroplets are subsequently ejected to form dots on the spaces between thefirst dots, thereby forming a continuous compartment.
 10. The process ofclaim 7, wherein a plurality of compartments of different sets aresimultaneously produced by ejection of liquid reagents from amultichannel jet head.
 11. The process of claim 7, wherein a volume ofeach droplet is 20 to 2000 picoliters.